PROJECT SUMMARY The primary objective of this proposal is to couple a) mechanistic insight relating differentiation outcomes to ECM engagement via intracellular signaling events triggered at the focal adhesion (FA), with b) 3D printing of ECM and ECM-associated proteins as a means to direct cell distribution with maturation and thereby enable fabrication of thick, functional cardiac tissue. The proposal is significant as it has the potential to generate replacement tissues and even heart grafts for individuals suffering from acute and chronic injury to the heart. It is the first of its kind to address the conundrum of the apparent uniformity of the focal adhesion relative to the myriad of different ECM/integrin combinations and the corresponding variety of cell behaviors that emerge from ECM engagement. It does so by proposing that elements of the FA, namely integrin linked kinase (ILK) and associated phosphatase, act as sensitive rheostats that can be co-opted to yield desired behavior. Here, the desired behavior is cardiac differentiation, and the innovation is the utilization of optimized ECM formulations as bioinks to create 3D cardiac tissue mimics (3DCTM) capable of directing cell distribution of multiple cell types with differentiation. This concept is feasible as the Ogle laboratory has long-studied the biochemistry of the ECM and stem cell behaviors associated with ECM engagement, and the McAlpine laboratory has focused on 3D printing of functional materials for a range of applications, from biological to electronic and the merger of these materials. These groups will also interface with expertise of the Kamp lab with respect to their recent generation of induced cardiac progenitor cells (iCPCs) to populate the 3DCTM, the Provenzano lab to assist with molecular mechanisms associated with FA formation, the Garry lab to assist with bioreactor implementation, the Zhang lab to add cardiovascular physiology expertise, and the Talkachova lab to assist with optical imaging to assess function of the 3DCTM and core facilities for mass spectrometry of ECM components and gene editing tools for modulating ILK activity. Together, this expertise will be funneled toward meeting the primary objective of the proposal via the following aims: 1) determine whether activation of integrin-linked kinase (ILK) of focal adhesions or costameres couples integrin activation to ?-catenin activation via GSK3? to enable expression of genes associated with cardiomyocyte specification, 2) use 3D ECM-based model systems to identify ECM formulations supportive of endothelial differentiation and assess ILK dependence, and 3) use ECM-based 3D printing to modulate differentiation of cardiac cell types spatially in a cardiac tissue mimic. The motivation for this concept was based on extensive literature search, and results from our own experimentation; the approach was designed to insure that the interpretations of the results are subject to minimal bias and the hypotheses posed are truly tested.